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Dual-target dsRNA (double-strand Ribonucleic Acid) as well as synthetic method and application thereof

A synthesis method and target technology, applied in the application of binary target dsRNA, in the field of binary target dsRNA synthesis, can solve problems such as environmental pollution, low lethality, energy waste, etc., achieve environmental friendliness, reduce production costs, and raw material costs low effect

Inactive Publication Date: 2018-01-09
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the deficiencies in the prior art, the present invention provides a dual-target dsRNA synthesis method and its application, which solves the problems of low lethality, environmental pollution and energy waste in existing methods for preventing and controlling pine wood nematode, and provides a new option for it

Method used

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  • Dual-target dsRNA (double-strand Ribonucleic Acid) as well as synthetic method and application thereof
  • Dual-target dsRNA (double-strand Ribonucleic Acid) as well as synthetic method and application thereof
  • Dual-target dsRNA (double-strand Ribonucleic Acid) as well as synthetic method and application thereof

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Embodiment 1

[0045] The synthetic method of binary target dsRNA (ras-mapk-dsRNA), the steps are as follows:

[0046] 1. Preparation of pine xylophilus cDNA

[0047] Use TRIZOL Reagent to extract total RNA, the steps are as follows:

[0048] 1) The ddH2O, centrifuge tubes and tips used in the RNA extraction process were treated with 0.1% DEPC.

[0049] 2) Collect pine wood nematodes in a 1.5ml centrifuge tube, quick-frozen in liquid nitrogen, add 200 μl Trizol, grind thoroughly with a micro tissue homogenizer, then add 800 μl Trizol, mix well and let stand at room temperature for 5 minutes.

[0050] 3) Add 200 μl of chloroform, shake fully, place at room temperature for 3 minutes, and centrifuge at 12000 rpm at 4°C for 15 minutes.

[0051] 4) Pipette the supernatant into another new 1.5ml centrifuge tube, add 550 μl of isopropanol, mix well, let stand at room temperature for 10 minutes, and centrifuge at 12000 rpm at 4°C for 10 minutes.

[0052] 5) Discard the supernatant, add 1ml 75% et...

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Abstract

The invention discloses dual-target dsRNA (double-strand Ribonucleic Acid) as well as a synthetic method and application thereof. The synthetic method is characterized by amplifying an MAPK (Mitogen-activated Protein Kinase) gene segment by taking cDNA (complementary Deoxyribonucleic Acid) of bursaphelenchus xylophilus as a template and MK-28i-R and MK-28i-F as primers, amplifying an RAS gene segment by taking RAS-28i-F and RAS-28i-R as primers, respectively connecting MAPK and RAS between dual T7 promoters of a vector LITMUS-28i through enzyme digestion and ligation, and constructing a recombinant vector LITMUS-28i-RAS-MAPK; then carrying out PCR (Polymerase Chain Reaction) amplification by using the recombinant vector as a template and T7 as a primer; synthesizing RAS-MAPK-dsRNA by taking a PCR amplification product as a template under the catalytic action of T7 RNA polymerase. The RAS-MAPK-dsRNA synthesized by the invention has a good prevention and control effect on the bursaphelenchus xylophilus, is environmentally friendly, convenient and quick, and has wide application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of a binary target dsRNA and a method for synthesizing the binary target dsRNA. Background technique [0002] The pine wood nematode (Bursaphelenchus xylophilus) belongs to the phylum Vermiformes, the class Nematodes, the order Ceromidae, the superfamily Slyceridae, and the genus Umbrella. When it infects pine trees, it will cause the death of pine trees, and it mainly harms masson pine and black pine. After being infected with the disease, in summer and autumn, the leaves lose water and turn yellow or reddish brown. Because the disease has the characteristics of fast onset and difficult to control, it is compared to the "cancer" of pine trees. As of January 2017, there were 243 county-level pine wood nematode epidemic areas in 15 provinces (cities) in my country. [0003] B. xylophilus is mainly transmitted by monochamus, and it has been reported that ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A01P5/00
Inventor 王殿东李娟何传平王紫薇
Owner YANGTZE NORMAL UNIVERSITY
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